1. Field of the Invention
The invention relates to electrophoretic separations of analytes and more particularly, to a method, an electrolyte composition, and a kit for independently altering the pH and mobility of ionic components of the composition. Further, the invention relates to a method, an electrolyte composition, and a kit for matching the mobility of ionic components of the composition with that of an analyte without altering, i.e., maintaining, the composition's pH.
2. Description of Related Art
Modern electrophoresis is a powerful approach to the separation and analysis of charged analytes, especially biopolymers. Separation is based on different electrophoretic mobilities of the analytes which, in turn, depend on charge densities. Electrophoretic migration may occur either in an open-robe fused silica capillary as in capillary zone electrophoresis (CZE) or in a supporting medium, such as in gel electrophoresis (GE).
Electrophoresis has been used to separate ions on the basis of their differential migration in an electric field. In addition to differences in mobility, the quality of these charge to volume ratio-based separations may also depend on the amount of band broadening the migrating analytes experience. The absolute ionic mobility, .mu..sup.0, is defined as the average velocity of an ion per unit of electric field strength at infinite dilution. This absolute ionic mobility is a characteristic constant for every ionic species in a certain solvent and is proportional to the equivalent conductance at zero concentration. The effective mobility of an ionic species is related to the absolute mobility. Corrections are made for influences such as the electrophoretic retardation and the relaxation effect. Thus, the effective mobility of an ionic species depends on several factors such as the ionic radius, solvation, dielectric constant and viscosity of the solvent, shape and charge of the ion, pH, degree of dissociation and temperature. Under certain conditions, band broadening in capillary electrophoresis is caused only by longitudinal diffusion. Generally, however, band broadening is larger than the broadening caused by diffusion alone. This may be due to the combined dispersion effects of injection, local temperature gradients, electroosmosis, and the heterogeneity of the electric field strength inside the capillary, i.e., electromigration dispersion. Of these factors, electromigration dispersion is normally the most important additional band broadening mechanism.
As noted above, electrophoretic separation of two analytes may only occur if the electrophoretic mobilities of the two components (.mu..sub.a1 and .mu..sub.a2) are different. Often the term separation selectivity, .alpha., which is the ratio of the two electrophoretic mobilities (.alpha.=.mu..sub.1a /.mu..sub.a2), is used to characterize the extent of this dissimilarity. The more different the value of .alpha. from unity, the easier it is to accomplish the separation. However, maximization of separation selectivity alone does not guarantee success in electrophoretic separations. An analyst should ensure that the bands of separated analytes remain narrow and do not mix with each other. The ability of the system to maintain narrow bands is characterized by the term separation efficiency. Separation efficiency may be limited only by the diffusive mixing, e.g., natural mixing due to concentration gradients in the separated bands. However, if the effective electrophoretic mobilities of the components of the electrolyte composition are significantly different from those of the analyte and the transfer number of the analytes is much larger than zero, an additional efficiency loss, brought about by electromigration dispersion (or mobility mismatch), may occur due to the distortion of the homogeneity of the electric field in the separation medium. Even mild electromigration dispersion may denigrate the separation.
The effects of electromigration dispersion may be minimized by using an electrolyte composition in which the transfer number of the analyte is about zero. However, due to insufficient detection sensitivity, this is often difficult or impossible to achieve. Another approach is to select buffers whose mobility closely matches the mobilities of the analytes. However, this approach also may fail because the mobility of the analytes and the buffer components vary to different degrees as the composition of the electrolyte composition is changed, e.g., as its pH is changed, or as additional secondary chemical equilibria-inducing agents, such as cyclodextrins, are added. The problem of mobility mismatch is further aggravated by the fact that the secondary chemical equilibria-inducing agents, such as cyclodextrins, may reduce the mobilities of the analytes by as much as 50 to 90%, and buffer components with such low mobilities in all of the useful pH ranges are not available.